Chlamydia infections are a cause of blindness worldwide and a major cause of sexually transmitted disease in the US. Greater understanding of protective immunity to Chlamydia at mucosal surfaces is urgently required if an effective vaccine is to become a reality. Our application will examine fundemental aspects of protective CD4 T cells during infection of the female reproductive tract mucosa. Our experimental approach is unique in that it utilizes a natural route of infection, uses an established model where CD4 T cells participate in bacterial clearance, and allows for direct visualization of endogenous Chlamydia-specific CD4 T cells using reagents that were recently developed in our laboratory. Our application specifically proposes to, (i) examine CD4 T cell heterogeneity and the contribution of these heterogeneous responses to protection and pathology, (ii) examine whether antibiotic-treatment adversely affects the development of protective TRM cells in the reproductive tract mucosa, (iii) determine whether the use of flagellin can enhance moderate protective immunity elicited by the single antigen PmpG1. Understanding each of these issues will increase our knowledge of Chlamydia-specific CD4 T cell biology and may be vitally important for the generation of a new Chlamydia vaccine.